Long terminal repeat (LTR) retrotransposons are mobile DNA sequences that ubiquitously exist in eukaryotic genomes. They replicate themselves in the genome by copy-paste mechanism with RNA as medium. In higher plants, many active LTR retrotransposons have been applied to analyze molecular marker technology, genetic tagging, insertion mutation and gene function. Here, we systematically review the characteristics of plant active LTR retrotransposons, including their structures, copy numbers and distributions. We further analyzed the gag (group-specific antigen) and pol (polymerase) sequence features of different plants active LTR retrotransposons and the distribution patterns of the cis-acting elements in LTR regions. The results show that autonomous active LTR retrotransposons must contain LTR regions and code Gag, Pr, Int, Rt, Rh proteins. Both LTR regions are highly homologous with each other and contain many cis-regulatory elements; RVT and RNase_H1_RT domain are essential for Rt and Rh protein respectively. These results provide the basis for subsequent identification of plant active LTR retrotransposons and their functional analysis.
Genome, Plant ; Mutagenesis, Insertional ; Plants ; genetics ; Retroelements ; Terminal Repeat Sequences

With the progess in studying gene structure and function of foot and mouth disease virus (FMDV), FMDV can express exogenous genes in different sites. Through transforming and modifying FMDV can achieve different application purposes such as improving virus titer, introducing tag, improving immune responses, and reducing pathogenicity. From the perspective of FMDV receiving inserted exogenous gene, this paper mainly describes the latest relevant developments of FMDV's expression to exogenous gene.
Foot-and-Mouth Disease Virus ; genetics ; Genetic Engineering ; Mutagenesis, Insertional

Hypoparathyroidism, sensorineural deafness, and renal dysgenesis syndrome is an autosomal dominant disease caused by mutations in the GATA3 gene on chromosome 10p15. We identified a patient diagnosed with hypoparathyroidism who also had a family history of hypoparathyroidism and sensorineural deafness, present in the father. The patient was subsequently diagnosed and found to be a heterozygote for an insertion mutation c.255_256ins4 (GTGC) in exon 2 of GATA3. His father was also confirmed to have the same mutation in GATA3.
Deafness* ; Exons ; Fathers ; Hearing Loss, Sensorineural ; Heterozygote ; Humans ; Hypoparathyroidism* ; Mutagenesis, Insertional

OBJECTIVETo investigate the genetic defect and its mechanism in a patient with congenital afibrinogenemia.

METHODSThe plasma fibrinogen activity and antigen of the patient was determined using the Clauss method and immuno-nephelometric assay, respectively. Genomic DNA was isolated from peripheral blood of the proband and his related family members. All exons and exon-intron boundaries of the three fibrinogen genes (FGA, FGB, FGG) were amplified by PCR followed by direct sequencing. Thrombin fibrin aggregation curve were detected in the plasma of the patient. Wild-type and mutation type fibrinogen vectors were constructed, and then transfected into COS-7 cells. The wild-type and mutant proteins from the culture media and cell lysates were tested by Western blot and ELISA.

RESULTSAPTT, PT, TT were significantly longer in the proband. Plasma fibrinogen activity and antigen of the patient could not be detected using the Clauss method and immuno-nephelometry, respectively. Gene analysis revealed that a novel homozygous GTTT insertion between nucleotides 2833 and 2834 in FGB exon 2 in the proband. The proband's father, mother, brother and son were heterozygous. The polymerization curves of the patient did not show a lag phase or final turbidity, compared with the normal controls. Western blot analysis showed the lack of complete half-molecules of the fibrinogen molecule and fibrinogen in patient's plasma under non-reducing conditions. It also could not detect the truncated Bβ chain under reducing conditions. Abnormal fibrinogen molecule (molecule weight>340 000) were found in transfected COS-7 cells by Western blot, which indicated that the mutation caused the abnormal intracellular fibrinogen molecule assembly. The fibrinogen band was absent in culture media transfected by the mutation. Fibrinogen levels of mutant fibrinogen were no significant different from those of wild-type fibrinogen in cell lysates by ELISA analysis [(2.47 ± 0.30) μg/ml vs (2.65±0.60) μg/ml, P=0.0889]; However, the levels of the mutant fibrinogen were statistically significant lower than those of wild type fibrinogen in culture media [(0.01 ± 0.01) μg/ml vs (3.80±0.80) μg/ml, P=0.0001].

CONCLUSIONCongenital afibrinogenemia was caused by this frameshift mutation in exon 2 of FGB. This novel mutation impaired fibrinogen assembly and secretion.

Afibrinogenemia ; congenital ; etiology ; genetics ; Fibrinogen ; genetics ; Frameshift Mutation ; Humans ; Male ; Mutagenesis, Insertional ; Pedigree ; Young Adult

A 38-year-old Korean man was diagnosed with hereditary sensory and autonomic neuropathy (HSAN) type 2 because of his chronic sensory neuropathy and progressive acro-mutilation. Genetic analysis revealed that he was a compound heterozygous for two novel mutations in the HSN2 gene including a nonsense mutation (Gln73X) and a 1-bp insertion mutation (Asp379fsX1). To our knowledge, this is the first report of a genetically confirmed case of HSAN type 2 in the Asian population and supports the genetic homogeneity of this rare disease.
Adult ; Asian Continental Ancestry Group ; Codon, Nonsense ; Hereditary Sensory and Autonomic Neuropathies ; Humans ; Mutagenesis, Insertional ; Rare Diseases

Insertional mutagenesis is a widely used method to determine the function(s) of a gene. To study the function(s) of the gene nsdAmgh in Streptomyces roseoflavus, a homologous recombination vector pSRNA2500 was structured in this paper. The recombination donor vector was then transformed into Strempomyces roseoflavus strain Men-myco-93-63 by conjugative transfer. The transformants were subjected to selection under the pressure of high temperature and appropriate antibiotics. As a result, several disrupted mutants of nsdAmgh gene, with a phenotype of Am(s)Km(r), were isolated and verified using PCR and Dot-blotting and Southern blotting hybridization methods. Functional analysis showed that the disrupted mutants of nsdAmgh had a two-fold higher inhibition against Verticillium dahlia Kleb than that of the wild strain Men-myco-93-63, which all will provide a new study route for future research about positive and negative regulator in Men-myco-93-63.
Genes, Bacterial ; Genetic Vectors ; Mutagenesis, Insertional ; Polymerase Chain Reaction ; Streptomyces ; genetics

DNA Damage ; Influenza A Virus, H1N1 Subtype ; Influenza, Human/*genetics ; Mutagenesis, Insertional/*genetics

OBJECTIVETo screen and identify zebrafish mutants with erythropoiesis defects by N-ethyl-N-nitrosourea (ENU) mutagenesis and large-scale forward genetic screening using beta e 1 as the marker.

METHODSThe chemical mutagen ENU was used to treat healthy wild-type male fish (AB strain, F0). The surviving ENU-treated fish were mated with wild-type female fish to generate F1, and further F2 family was generated by F1 family intercross. The adult F2 fish were intercrossed within each F2 family and the resulting F3 embryos from each crossing were subjected to whole mount in situ hybridization (WISH) with the beta e 1 probe. Mutagenesis was performed by treating the male zebrafish with ENU to induce mutations in pre-meiotic germ cells to generate the founders, which were outcrossed to obtained the F1 fish. The F1 fish from different founders were mated to generate the F2 families. F3 embryos from the sibling cross in the F2 family were examined by whole mount in situ hybridization using beta e 1-globin probe. The putative mutants were then characterized with different hematopoiesis markers.

RESULTS AND CONCLUSIONWe identified 4 beta e 1-deficient mutants with erythropoiesis defects, including two with specific erythiod lineage defects and two with concurrent lymphopoiesis defects.

Animals ; Erythropoiesis ; genetics ; Ethylnitrosourea ; Female ; Gene Expression Regulation, Developmental ; Male ; Mutagenesis, Insertional ; Mutation ; Zebrafish ; genetics

Ralstonia solanacearum strain PRS-84 used in this study was isolated from diseased Pogostemon cablin plants in our previous study.The competent cells of R.solanacearum strain PRS-84 were transformed by electroporation with Tn5 transposon and then were plated on TTC agar plates containing kanamycin to select for kanamycin-resistant colonies.The detection of kanamycin-resistant gene in kanamycin-resistant colonies was performed by PCR.Further,the flanking fragments of Tn5 transposon insertion site in the mutants were amplified by inverse PCR,and the flanking fragments were sequenced and analyzed.The results indicated that the kanamycin-resistant colonies were obtained in the transformation experiment of R.solanacearum strain PRS-84 by electroporation with Tn5 transposon.A specific band of approximately 700 bp was amplified by PCR from kanamycin-resistant colonies.The flanking sequences of Tn5 transposon insertion site in the transformants were obtained by inverse PCR.After sequencing and sequence analysis of Tn5 transposon insertion site in mutants,we preliminarily speculated that the Tn5 transposon inserted in the typ A gene,rec O gene and gid A gene in three mutants,respectively.A random mutagenesis system of R.solanacearum strain PRS-84 by electroporation with Tn5 transposon has been established,and the Tn5 insertion mutants have been obtained.This study might facilitate the creation of mutant library and the discovery of the virulence gene of R.solanacearum isolated from P.cablin.
DNA Transposable Elements ; Electroporation ; Genes, Bacterial ; Mutagenesis, Insertional ; Pogostemon ; microbiology ; Ralstonia solanacearum ; genetics ; Virulence

Epidermal growth factor receptor exon 20 insertion (EGFR ex20ins) is one of the earliest driver gene activation mutations in non-small cell lung cancer (NSCLC). However, due to the unique structure of protein variation caused by this mutation, most patients with EGFR ex20ins mutation (except A763_Y764insFQEA) have poor response to the launched first/second/third generation EGFR-tyrosine kinase inhibitors (EGFR-TKIs). With the successive approval of new specific targeted drugs for EGFR ex20ins in Food and Drug Administration (FDA) and other national regulatory agencies, the development and clinical research of targeted drugs for EGFR ex20ins in China have also developed rapidly and Mobocertinib has been approved recently in China. It is worth noting that EGFR ex20ins is a variant type with strong molecular heterogeneity. How to detect it comprehensively and accurately in clinical practice, so as to enable more patients to benefit from targeted therapy, is a very important and urgent problem to be solved. This review introduces the molecular typing of EGFR ex20ins, then discusses the importance of EGFR ex20ins detection and the differences of various detection methods, and summarizes the research and development of new drugs progress of EGFR ex20ins, in order to optimize the diagnosis and treatment path of EGFR ex20ins patients by selecting accurate, rapid and appropriate detection methods, so as to improve the clinical benefits of the patients.
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United States ; Humans ; Carcinoma, Non-Small-Cell Lung ; Mutagenesis, Insertional ; Lung Neoplasms ; ErbB Receptors ; Exons